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Abstract:

Disclosed, among other things, are primers containing certain modified
nucleobases in the 3' terminal region of the primers that provide reduced
formation of primer-dimers during amplification reactions, and various
methods of use thereof.

Claims:

1-98. (canceled)

99. A method of DNA amplicon fragment analysis comprising: i) annealing
an oligonucleotide primer to a denatured DNA template such that, the
oligonucleotide primer anneals to a complementary oligonucleotide
sequence on a strand of the denatured DNA template to form a
primer-template complex, wherein the oligonucleotide primer comprises at
least one modified purine nucleobase comprising the structure:
##STR00012## wherein R1 is selected from hydrogen, halogen,
fluorine, chlorine, bromine, iodine, azido, nitro, cyano, unsubstituted
or substituted amino, C1-C6 alkyl, C1-C6 alkynyl,
C1-C6 substituted alkynyl, unsubstituted or substituted
phenylalkynyl, and unsubstituted or substituted aryl; at least one said
modified purine nucleobase is no more than 3 nucleotides from the 3'
terminus of the oligonucleotide primer; and the oligonucleotide primer is
extendable at its 3'-end; ii) extending the primer portion of the
primer-template complex in the presence of extendable nucleotide
triphosphates and non-extendable nucleotide triphosphates to form one or
more DNA amplicon fragments; and iii) detecting the DNA amplicon
fragments.

100. The method of claim 99, wherein the at least one modified purine
nucleobase is no more than 2 nucleotides from the 3' terminus of the
oligonucleotide primer.

101. The method of claim 99, wherein the at least one modified purine
nucleobase is no more than 1 nucleotides from the 3' terminus of the
oligonucleotide primer.

102. The method of claim 99, wherein the at least one modified purine
nucleobase is the 3' terminal nucleotide of the oligonucleotide primer.

107. The method of claim 106 further comprising detecting the DNA
amplicon fragments by laser-induced fluorescence.

108. The method of claim 105, comprising using a plurality of labeled
oligonucleotide primers wherein each labeled oligonucleotide primer is
labeled with a different and spectrally resolvable label.

109. The method of claim 108, wherein the DNA amplicon fragments are
formed using chain termination methods of DNA sequencing.

110. The method of claim 109 wherein the DNA amplicon fragments may be
identified by identifying terminal nucleotides in each of the DNA
amplicon fragment and wherein a correspondence is established between the
four possible terminal nucleotides and a set of spectrally resolvable
labels.

111. The method of claim 99, wherein the oligonucleotide primer
comprising the at least one said modified purine nucleobase comprises the
structure: ##STR00013##

112. The method of claim 99, wherein the oligonucleotide primer
comprising the at least one said modified purine nucleobase comprises the
structure: ##STR00014##

113. The method of claim 99, wherein the oligonucleotide primer
comprising the at least one said modified purine nucleobase comprises the
structure: ##STR00015##

114. A method of DNA amplicon fragment analysis comprising: i) annealing
an oligonucleotide primer to a denatured DNA template such that, the
oligonucleotide primer anneals to a complementary oligonucleotide
sequence on a strand of the denatured DNA template to form a
primer-template complex, wherein the oligonucleotide primer comprises at
least one modified purine nucleobase comprising the structure:
##STR00016## wherein R1 is selected from hydrogen, halogen,
fluorine, chlorine, bromine, iodine, azido, nitro, cyano, unsubstituted
or substituted amino, C1-C6 alkyl, C1-C6 alkynyl,
C1-C6 substituted alkynyl, unsubstituted or substituted
phenylalkynyl, and unsubstituted or substituted aryl; at least one said
modified purine nucleobase is no more than 3 nucleotides from the 3'
terminus of the oligonucleotide primer, and the oligonucleotide primer is
extendable at its 3'-end; ii) extending the primer portion of the
primer-template complex in the presence of deoxyribonucleic acids and
non-extendable ribonucleic acids to form one or more DNA amplicon
fragments; and iii) detecting the DNA amplicon fragments.

115. The method of claim 114, wherein the oligonucleotide primer
comprising the at least one said modified purine nucleobase comprises the
structure: ##STR00017##

116. The method of claim 114, wherein the oligonucleotide primer
comprising the at least one said modified purine nucleobase comprises the
structure: ##STR00018##

117. The method of claim 114, wherein the oligonucleotide primer
comprising the at least one said modified purine nucleobase comprises the
structure: ##STR00019##

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser.
No. 11/250,192, filed Oct. 12, 2005, which is a continuation of U.S.
patent application Ser. No. 11/106,021, filed Apr. 14, 2005, now
abandoned, which claims a priority benefit under 35 U.S.C. §119(e)
from U.S. Patent Application No. 60/562,388, filed Apr. 14, 2004, which
is incorporated herein by reference.

[0002] The present teachings relate to nucleic acid amplification, for
example, compounds and methods for application in the polymerase chain
reaction (PCR).

[0003] Detecting the presence of target nucleic acids plays an important
role in a variety for applications in diverse fields, including: medical
diagnostics, forensic science and genetic analysis. PCR is an example of
a nucleic acid amplification method that can provide a highly sensitive
means for detecting the presence of target nucleic acids by selective
amplification of a target nucleic acid sequence.

[0004] A significant problem with nucleic acid amplifications such as PCR
is the generation of non-specific amplification products. One example of
a non-specific amplification process that can be problematic in PCR
reactions is "primer-dimer" amplification. Primer-dimer amplification can
result when, for example, the 3' terminal region of a primer has some
degree of complimentarity with itself or another primer. Such primers can
hybridize to one another to form primer-dimers. Amplification of the
primer-dimer can then lead to primer-dimer amplicons that can in turn act
as templates for further amplification. One outcome of such a process
being a depletion of primers resulting in reduced sensitivity or even a
failure to amplify the intended target nucleic acid.

[0005] To complicate the problem, the addition of a large excess of
primers during PCR reactions allows even weak complimentarity at the 3'
terminal region to result in primer-dimer amplicons. As a result there is
a need to develop reagents and methods that suppress primer-dimer
formation in amplification reactions such as PCR.

[0006] It has now been found that, surprisingly, incorporation of certain
modified nucleobases in the 3' terminal region of primers can
significantly reduce the formation of primer-dimer amplicons during
amplification reactions.

[0007] In some embodiments, the present teachings provide polynucleotides
comprising at least one modified purine nucleobase of the structure

##STR00001##

wherein R1 is selected from hydrogen, halogen, fluorine, chlorine,
bromine, iodine, azido, nitro, cyano, unsubstituted or substituted amino,
C1-C6 alkyl, C1-C6 alkynyl, C1-C6
substituted alkynyl, unsubstituted or substituted phenylalkynyl, and
unsubstituted or substituted aryl, or R1 is selected from hydrogen,
halogen, fluorine, chlorine, bromine, iodine, C1-C6 alkyl,
C1-C6 alkynyl, and C1-C6 substituted alkynyl, such
that at least one said modified purine nucleobase is no more than 3
nucleotides from the 3' terminus of the primer. In some embodiments,
polynucleotides of the present invention are extendable at the 3'-end. In
some embodiments, at least one said modified purine nucleobase comprises
the structure

##STR00002##

[0008] In some embodiments, at least one said modified purine nucleobase
is no more than 2 nucleotides from the 3' terminus of the polynucleotide.
In some embodiments, at least one said modified purine nucleobase is no
more than 1 nucleotide from the 3' terminus of the polynucleotide. In
some embodiments, at least one said modified purine nucleobase is the 3'
terminal nucleobase of the polynucleotide. In some embodiments,
polynucleotides of the present teachings can be polynucleotide primers.
In some embodiments, polynucleotide primers of the present teachings can
include at least one of a detectable label, a quencher or a minor groove
binder, or a combination thereof.

[0009] In some embodiments, the present teachings provide for methods of
primer extension comprising, annealing a polynucleotide primer to a
denatured DNA template such that, the polynucleotide primer anneals to a
complementary polynucleotide sequence on a strand of the denatured DNA
template to form a primer-template complex, and extending the primer
portion of the primer-template complex to form a double stranded
amplicon, wherein the polynucleotide primer is a primer according to the
present teachings.

[0010] In some embodiments, the present teachings provide for methods of
primer extension comprising, after the step of extending, denaturing the
double stranded amplicon. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least one time. In some
embodiments, the steps of annealing, extending and denaturing can be
repeated at least 10 times. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least 20 times. In some
embodiments, the steps of annealing, extending and denaturing can be
repeated at least 30 times. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least 40 times.

[0011] In some embodiments, the present teachings provide for methods of
primer extension, wherein the extending takes place in the presence of
extendable nucleotide triphosphates and non-extendable nucleotide
triphosphates to form DNA amplicon fragments. In some embodiments the
method of primer extension comprises, detecting the DNA amplicon
fragments.

[0012] In some embodiments, the present teachings provide methods of
primer extension comprising: i) annealing a first polynucleotide primer
and a second polynucleotide primer to a first and second strand of a
denatured DNA template such that, the first polynucleotide primer anneals
to a complementary oligonucleotide sequence on the first strand of the
denatured DNA template and the second polynucleotide primer anneals to a
complementary oligonucleotide sequence on the second strand of the
denatured DNA template to form a first and a second primer-template
complex, and ii) extending the primer portion of at least one of the
first and second primer-template complex to form double stranded DNA
amplicon, where at least one of the first polynucleotide primer or the
second polynucleotide primer can be a polynucleotide according to the
present teachings.

[0013] In some embodiments, the present teachings provide methods of
primer extension comprising, prior to the step of annealing, forming a
mixture comprising a first polynucleotide primer, a second polynucleotide
primer, a DNA template, and other primer extension reagents. In some
embodiments, the present teachings provide methods of primer extension
comprising, after the step of forming but prior to the step of annealing,
denaturing the DNA template to form a first strand of denatured DNA
template and a second denatured DNA template. In some embodiments, the
present teachings provide methods of primer extension comprising, after
the step of extending, denaturing the double stranded DNA amplicon.

[0014] In some embodiments, the steps of annealing, extending and
denaturing the double stranded DNA amplicon can optionally be repeated
from 1-100 times. Optionally, the steps of annealing, extending and
denaturing the double stranded DNA amplicon can be repeated from 1-50
times. It will be understood that the present teachings encompass all
possible ranges for repeating the steps of annealing, extending and
denaturing the double stranded DNA amplicon between 1 and 100 times. That
is, the steps of annealing, extending and denaturing the double stranded
DNA amplicon can be repeated from 1 time up to 100 times and any number
of times in between. For example, the range, of 1-10 will be understood
to include all possible ranges using all integers between 1 and 10,
i.e.-1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In some embodiments, the steps of
annealing, extending and denaturing the double stranded DNA amplicon can
optionally be repeated greater than 1 time. In some embodiments, the
steps of annealing, extending and denaturing the double stranded DNA
amplicon can optionally be repeated greater than 10 times. In some
embodiments, the steps of annealing, extending and denaturing the double
stranded DNA amplicon can optionally be repeated greater than 20 times.
In some embodiments, the steps of annealing, extending and denaturing the
double stranded DNA amplicon can optionally be repeated greater than 30
times. In some embodiments, the steps of annealing, extending and
denaturing the double stranded DNA amplicon can optionally be repeated
greater than 40 times. In some embodiments, the steps of annealing,
extending and denaturing the double stranded DNA amplicon can optionally
be repeated greater than 50 times.

[0015] In some embodiments, the present teachings provide for methods of
primer extension comprising, prior to the step of extending the primer
portion, annealing a polynucleotide probe to a first or second strand of
a denatured DNA template such that, the polynucleotide probe anneals to a
complementary polynucleotide sequence on the first strand of the
denatured DNA template and/or the polynucleotide probe anneals to a
complementary oligonucleotide sequence on the second strand of the
denatured DNA template. In some embodiments, the polynucleotide probe
comprises at least one detectable label. In some embodiments, the
polynucleotide probe further comprises at least one of a quencher, a
minor groove binder or both. In some embodiments, the polynucleotide
probe can be a polynucleotide of the present teachings.

[0016] In some embodiments, the present teachings provide methods of
oligonucleotide ligation comprising, i) forming a complex comprising a
first and a second polynucleotide strand annealed to a DNA template such
that, the first polynucleotide strand anneals to a first complementary
polynucleotide sequence on the strand of the denatured DNA template and
the second polynucleotide strand anneals to a second complementary
polynucleotide sequence on the strand of the denatured DNA template,
wherein the second complementary polynucleotide sequence on the strand of
the denatured DNA template is located 5' to the first complementary
polynucleotide sequence on the strand of the denatured DNA template, and
ii) forming a stable covalent bond between the first and second
polynucleotide strands, wherein at least one of the first polynucleotide
strand or the second polynucleotide strand is a polynucleotide of the
present teachings.

[0017] In some embodiments, the present teachings provide for methods for
detecting a target polynucleotide sequence comprising, (a) reacting a
target polynucleotide strand with a first probe pair comprising (i) a
first polynucleotide probe containing a sequence that is complementary to
a first target region in the target strand and (ii) a second
polynucleotide probe comprising a sequence that is complementary to a
second target region in the target strand, wherein the second region is
located 5' to the first region and overlaps the first region by at least
one nucleotide base, under conditions effective for the first and second
probes to hybridize to the first and second regions in the target strand,
respectively, forming a first hybridization complex, (b) cleaving the
second probe in the first hybridization complex, to form a second
hybridization complex comprising the target strand, the first probe, and
a first fragment of the second probe having a 5' terminal nucleotide
located immediately contiguous to a 3' terminal nucleotide of the first
probe, (c) ligating the first probe to the hybridized fragment of the
second probe to form a first ligated strand hybridized to the target
strand, (d) denaturing the first ligated strand from the target strand,
and (e) performing one or more additional cycles of steps (a) through
(d), with the proviso that in the last cycle, step (d) is optionally
omitted, wherein at least one of the first probe, the second probe or
both is a polynucleotide comprising at least one modified purine
nucleobase comprising the structure

##STR00003##

wherein R1 is selected from hydrogen, halogen, fluorine, chlorine,
bromine, iodine, azido, nitro, cyano, unsubstituted or substituted amino,
C1-C6 alkyl, C1-C6 alkynyl, C1-C6
substituted alkynyl, unsubstituted or substituted phenylalkynyl, and
unsubstituted or substituted aryl, or wherein R1 is selected from
hydrogen, halogen, fluorine, chlorine, bromine, iodine, C1-C6
alkyl, C1-C6 alkynyl, and C1-C6 substituted alkynyl,
such that at least one said modified purine nucleobase is no more than 3
nucleotides from the 3' terminus of the first polynucleotide probe or the
second polynucleotide probe or both, and the first polynucleotide probe
is extendable at its 3'-end. In some embodiments, at least one said
modified purine nucleobase comprises the structure

##STR00004##

[0018] In some embodiments, at least one said modified purine nucleobase
is no more than 2 nucleotides from the 3' terminus of the first
polynucleotide probe, the second polynucleotide probe or both. In some
embodiments, at least one said modified purine nucleobase is no more than
1 nucleotides from the 3' terminus of the first polynucleotide probe, the
second polynucleotide probe or both. In some embodiments, the modified
purine nucleobase is the 3' terminal nucleotide of the first
polynucleotide probe, the second polynucleotide probe or both.

[0019] In some embodiments, the first polynucleotide probe, the second
polynucleotide probe or both comprise at least one of a detectable label,
a quencher or a minor groove binder, or any combination thereof. In some
embodiments, the first region overlaps the second region by one
nucleotide base. In some embodiments, the 5' end of the first probe
terminates with a group other than a nucleotide 5' phosphate group. In
some embodiments, the 5' end of the first probe terminates with a
nucleotide 5' hydroxyl group. In some embodiments, the 5' end of the
second probe terminates with a group other than a nucleotide 5' phosphate
group. In some embodiments, the 5' end of the second probe terminates
with a nucleotide 5' hydroxyl group. In some embodiments, the 3' end of
the second probe terminates with a group other than a nucleotide 3'
hydroxyl group. In some embodiments, the 3' end of the second probe
terminates with a nucleotide 3' phosphate group.

[0020] In some embodiments, the present teachings provide methods for
detecting a target polynucleotide sequence comprising, (a) reacting a
target-complementary strand with a second probe pair comprising (i) a
third polynucleotide probe containing a sequence that is complementary to
a first region in the target-complementary strand and (ii) a fourth
polynucleotide probe containing a sequence that is complementary to a
second region in the target-complementary strand, wherein the second
region is located 5' to the first region and overlaps the first region by
at least one nucleotide base, under conditions effective for the for the
third and fourth probes to hybridize to the first and second regions in
the target-complementary strand, respectively, forming a third
hybridization complex, (b) cleaving the fourth probe in the second
hybridization complex, to form a forth hybridization complex comprising
the target-complementary strand, the third probe, and a first fragment of
the forth probe having a 5' terminal nucleotide located immediately
contiguous to a 3' terminal nucleotide of the third probe, (c) ligating
the third probe to the hybridized fragment of the fourth probe to form a
second ligated strand hybridized to the target-complementary strand, (d)
denaturing the second ligated strand from the target-complementary
strand, and (e) performing one or more additional cycles of steps (a)
through (d), with the proviso that in the last cycle, step (d) is
optionally omitted. In some embodiments, at least one of the third probe
or the forth probe or both is a polynucleotide comprising at least one
modified purine nucleobase comprising the structure

##STR00005##

wherein R1 is selected from hydrogen, halogen, fluorine, chlorine,
bromine, iodine, azido, nitro, cyano, unsubstituted or substituted amino,
C1-C6 alkyl, C1-C6 alkynyl, C1-C6
substituted alkynyl, unsubstituted or substituted phenylalkynyl, and
unsubstituted or substituted aryl, or wherein R1 is selected from
hydrogen, halogen, fluorine, chlorine, bromine, iodine, C1-C6
alkyl, C1-C6 alkynyl, and C1-C6 substituted alkynyl,
such that at least one said modified purine nucleobase is no more than 3
nucleotides from the 3' terminus of the third probe or the forth probe or
both, and the third polynucleotide probe is extendable at its 3'-end. In
some embodiments, at least one said modified purine nucleobase comprises
the structure

##STR00006##

[0021] In some embodiments, at least one said modified purine nucleobase
is no more than 2 nucleotides from the 3' terminus of the first
polynucleotide probe, the second polynucleotide probe or both. In some
embodiments, at least one said modified purine nucleobase is no more than
1 nucleotide from the 3' terminus of the first polynucleotide probe, the
second polynucleotide probe or both. In some embodiments, said modified
purine nucleobase can be the 3' terminal nucleotide of the first
polynucleotide probe, the second polynucleotide probe or both. In some
embodiments, the first polynucleotide probe, the second polynucleotide
probe or both can comprise at least one of a detectable label, a quencher
or a minor groove binder, or any combination thereof. In some
embodiments, the 5' end of the third probe optionally terminates with a
group other than a nucleotide 5' phosphate group. In some embodiments,
the 5' end of the third probe optionally terminates with a nucleotide 5'
hydroxyl group. In some embodiments, the 5' end of the fourth probe
optionally terminates with a group other than a nucleotide 5' phosphate
group. In some embodiments, the 5' end of the fourth probe optionally
terminates with a nucleotide 5' hydroxyl group. In some embodiments, the
5' ends of the first, second, third and fourth probes optionally
terminate with a group other than a nucleotide 5' phosphate group. In
some embodiments, the 3' end of the fourth probe optionally terminates
with a group other than a nucleotide 3' hydroxyl group. In some
embodiments, the 3' end of the fourth probe optionally terminates with a
nucleotide 3' phosphate group. In some embodiments, at least one of the
probes contains a detectable label. In some embodiments, the label can be
a fluorescent label. In some embodiments, the label can be a radiolabel.
In some embodiments, the label can be a chemiluminescent label. In some
embodiments, the label can be an enzyme. In some embodiments, at least
one of the first probe and the third probe contains a detectable label.
In some embodiments, each of the first probe and third probe contains a
detectable label. In some embodiments, the detectable labels on the first
probe and third probe are the same. In some embodiments, at least one of
the second probe and the fourth probe contains a detectable label. In
some embodiments, each of the second probe and the fourth probe contains
a detectable label. In some embodiments, the second probe and fourth
probe contain the same detectable label. In some embodiments, said
cleaving produces a second fragment from the second probe which does not
associate with the second hybridization complex, and the method further
includes detecting said second fragment from the second probe. In some
embodiments, said cleaving produces a second fragment from the forth
probe which does not associate with the forth hybridization complex, and
the method further includes detecting said second fragment from the forth
probe. In some embodiments, at least one of the second probe and the
fourth probe contains both (i) a fluorescent dye and (ii) a quencher dye
which is capable of quenching fluorescence emission from the fluorescent
dye when the fluorescent dye is subjected to fluorescence excitation
energy, and said cleaving severs a covalent linkage between the
fluorescent dye and the quencher dye in the second probe and/or fourth
probe, thereby increasing an observable fluorescence signal from the
fluorescent dye. In some embodiments, the second probe and the fourth
probe each contain (i) a fluorescent dye and (ii) a quencher dye.

[0022] In some embodiments, methods of the present teachings for detecting
target polynucleotide sequences method further include detecting both
second fragments. In some embodiments, the second fragment comprises one
or more contiguous nucleotides substantially non-complementary to the
target strand. In some embodiments, the one or more contiguous
nucleotides comprise 1 to 20 nucleotides.

[0023] In some embodiments, methods of the present teachings for detecting
target polynucleotide sequences further include immobilizing the second
fragment on a solid support. In some embodiments, methods of the present
teachings for detecting target polynucleotide sequences further include
subjecting the second fragment to electrophoresis. In some embodiments,
methods of the present teachings for detecting target polynucleotide
sequences further include detecting the second fragment by mass
spectrometry. In some embodiments, methods of the present teachings for
detecting target polynucleotide sequences comprise detecting the second
fragment after the last cycle. In some embodiments, methods of the
present teachings for detecting target polynucleotide sequences comprise
detecting the second fragment during or after a plurality of cycles. In
some embodiments, methods of the present teachings for detecting target
polynucleotide sequences comprise detecting the second fragment during
all of the cycles.

[0024] In some embodiments, methods of the present teachings for detecting
target polynucleotide sequences further include detecting the first
hybridization complex, the second hybridization complex, or both, after
at least one cycle. In some embodiments, the method further includes
detecting the third hybridization complex, the fourth hybridization
complex, or both, after at least one cycle. In some embodiments, the
method further includes detecting the first ligated strand, the second
ligated strand, or both, after at least one cycle. In some embodiments,
the detecting comprises an electrophoretic separation step.

[0025] In some embodiments, the present teachings provide for methods of
fragment analysis comprising: i) annealing an oligonucleotide primer to a
denatured DNA template such that, the oligonucleotide primer anneals to a
complementary oligonucleotide sequence on a strand of the denatured DNA
template to form a primer-template complex, ii) extending the primer
portion of the primer-template complex in the presence of
deoxyribonucleic acids and non-extendable ribonucleic acids to form DNA
amplicon fragments, and iii) detecting the DNA amplicon fragments, where
the oligonucleotide primer is an polynucleotide of the present teachings.

[0026] Scheme 1 below illustrates an exemplary polynucleotide comprising a
plurality, (x), of nucleotides, "N", that may define a desired nucleotide
sequence, wherein the subscripts 1, 2, 3 . . . x refer to the position of
the nucleotide in the primer relative to the 3' end, and " . . . "
indicates the possibility of one or more additional nucleotides between
Nx and N7.

5'-Nx . . . N7N6N5N4N3N2N1-3'
Scheme 1

Thus, N1 is located at the 3' terminus of the exemplary
polynucleotide, and can be referred to as the 3' terminal nucleotide of
the exemplary polynucleotide. Similarly, N2 is located at the second
nucleotide position, and can be referred to as being 1 nucleotide from
the 3' terminus. Similarly, N3 is located at the third nucleotide
position, and can be referred to as being 2 nucleotides from the 3'
terminus. Similarly, N4 is located at the fourth nucleotide
position, and can be referred to as being 3 nucleotides from the 3'
terminus. It is believed that the effect of reduced primer-dimer
formation that results from incorporation of nucleobases of the present
teachings into primers will decrease in primers having no nucleotide of
the present teachings any nearer the 3'-terminus than about the N4
position.

[0027] As used herein, the terms oligonucleotide, polynucleotide and
nucleic acid are used interchangeably to refer to single- or
double-stranded polymers of DNA, RNA or both including polymers
containing modified or non-naturally occurring nucleotides. In addition,
the terms oligonucleotide, polynucleotide and nucleic acid refer to any
other type of polymer comprising a backbone and a plurality of
nucleobases that can form a duplex with a complimentary polynucleotide
strand by nucleobase-specific base-pairing. including, but not limited
to, peptide nucleic acids (PNAs) which are disclosed in, for example,
Nielsen et al., Science 254:1497-1500 (1991), bicyclo DNA oligomers
(Bolli et al., Nucleic Acids Res. 24:4660-4667 (1996)) and related
structures.

[0028] In some embodiments, polynucleotides of the present teachings can
comprise a backbone of naturally occurring sugar or glycosidic moieties,
for example, β-D-ribofuranose. In addition, in some embodiments,
modified nucleotides of the present teachings can comprise a backbone
that includes one or more "sugar analogs". As used herein, the term
"sugar analog" refers to analogs of the sugar ribose. Exemplary ribose
sugar analogs include, but are not limited to, substituted or
unsubstituted furanoses having more or fewer than 5 ring atoms, e.g.,
erythroses and hexoses and substituted or unsubstituted 3-6 carbon
acyclic sugars. Typical substituted furanoses and acyclic sugars are
those in which one or more of the carbon atoms are substituted with one
or more of the same or different --R, --OR, --NRR or halogen groups,
where each R is independently --H, (C1-C6) alkyl or
(C3-C14) aryl. Examples of unsubstituted and substituted
furanoses having 5 ring atoms include but are not limited to
2'-deoxyribose, 2'-(C1-C6)-alkylribose,
2'-(C1-C6)-alkoxyribose, 2'-(C5-C14)-aryloxyribose,
2',3'-dideoxyribose, 2',3'-dideoxy-ribose, 2'-deoxy-3'-haloribose,
2'-deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose,
2'-de-oxy-3'-aminoribose, 2'-deoxy-3'-(C1-C6)-alkylribose,
2'-deoxy-3'-(C1-C6)-alkoxyribose,
2'-deoxy-3'-(C5-C14), 3'-(C1-C6)-aryloxyribose
alkylribose-5'-triphosphate,
2'-deoxy-3'-(C1-C6)-alkylribose-5'-triphosphate,
2'-deoxy-3'-(C5-C14)
2'-deoxy-3'-(C1-C6)-alkoxyribose-5'-triphosphate,
-aryl-oxyribose-5'-triphosphate, 2'-deoxy-3'-haloribose-5'-triphosphate,
2'-deoxy-3'-aminoribose-5'-triphosphate,
2',3'-dideoxyribose-5'-triphosphate or
2',3'-dide-hydroribose-5'-triphosphate. Further sugar analogs include but
are not limited to, for example "locked nucleic acids" (LNAs), i.e.,
those that contain, for example, a methylene bridge between C-4' and an
oxygen atom at C-2', such as

[0029] In some embodiments, polynucleotides of the present teachings
include those in which the phosphate backbone comprises one or more
"phosphate analogs". The term "phosphate analog" refers to analogs of
phosphate wherein the phosphorous atom is in the +5 oxidation state and
one or more of the oxygen atoms are replaced with a non-oxygen moiety.
Exemplary analogs include, but are not limited to, phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate,
boronophosphates, and associated counterions, including but not limited
to H+, NH4+, Na+, Mg++ if such counterions are
present. Polynucleotides of the present teachings containing phosphate
analogs can comprise, for example, phosphorothioate linkages,
methylphosphonates and/or phosphoroamidates (see, Chen et al., Nucl Acids
Res., 23:2662-2668 (1995)). Combinations of polynucleotide linkages are
also within the scope of the present teachings.

[0030] In some embodiments, polynucleotides described herein can be
incorporated into PNA and DNA/PNA chimeras. Including, peptide nucleic
acids (PNAs, also known as polyamide nucleic acids), see, for example,
Nielsen et al., Science 254:1497-1500 (1991). PNAs contain heterocyclic
nucleobase units that are linked by a polyamide backbone instead of the
sugar-phosphate backbone characteristic of DNA and RNA. PNAs are capable
of hybridization to complementary DNA and RNA target sequences. Synthesis
of PNA oligomers and reactive monomers used in the synthesis of PNA
oligomers are described in, for example, U.S. Pat. Nos. 5,539,082;
5,714,331; 5,773,571; 5,736,336 and 5,766,855. Alternate approaches to
PNA and DNA/PNA chimera synthesis and monomers for PNA synthesis have
been summarized in, for example, Uhlmann, et al., Angew. Chem. Int. Ed.
37:2796-2823 (1998).

[0031] In some embodiments, polynucleotides of the present teachings can
range in size from a few nucleotide monomers in length, e.g. from 5 to
80, to hundreds or thousands of nucleotide monomers in length. For
example, polynucleotides of the present teachings can contain from 5 to
50 nucleotides, 5 to 30 nucleotides, or 5 to 20 nucleotides. When, in
some embodiments, polynucleotides of the present teachings contain, for
example, from 5 to 30 nucleotides, such a range includes all possible
ranges of integers between 5 and 30, for example 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 an
30 nucleotides in length. Whenever a polynucleotide is represented by a
sequence of letters, such as "ATGCCTG," it will be understood that the
nucleotides are in 5' to 3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine,
and T denotes thymidine, unless otherwise indicated. Additionally,
whenever a polynucleotide of the present teachings is represented by a
sequence of letters that includes an "X", it will be understood that the
"X" denotes a variable nucleotide monomer, where "X" is a nucleotide
monomer other than "A", "C", "G" or "T".

[0032] In some embodiments, polynucleotides of the present teachings can
serve as primers in amplification reactions. As used herein, "primer"
refers to a polynucleotide as defined herein having a 3' terminus that is
extendable by addition of one or more nucleotide monomers or by ligation
of a ligation probe.

[0033] In some embodiments, the present teachings provide for
polynucleotide primers comprising at least one nucleotide having a
nucleobase of the structure

##STR00008##

wherein R1 is selected from hydrogen, halogen, fluorine, chlorine,
bromine, iodine, C1-C6 alkyl, C1-C6 alkynyl, and
C1-C6 substituted alkynyl, at least one said modified purine
nucleobase is no more than 3 nucleotides from the 3' terminus and the
polynucleotide primer is extendable at the 3'-end.

[0034] As used herein, "substituted" as in "substituted alkynyl" means
that the moiety that is substituted (e.g., alkynyl moiety) comprises one
or more non-hydrogen substituents. Such substituents include, but are not
limited to, --F, --Cl, --Br, --CF3, amine, --OH, --CCl3, --CN,
--CHO, --CO2R, --SO3R, --PO3RR, --C(O)NRR and --NO2
where R can be any of --H, C1-C6 alkyl or C3-C10
aryl.

[0038] As used herein, unsubstituted amino means NH2, and substituted
amino means mono or di-substituted amino of the form NRaRb,
wherein Ra and Rb are each separately selected from --H,
C1-C6 alkyl, substituted C1-C6 alkyl, or together are
C1-C6 alkyl diradical chain or substituted C1-C6
alkyl diradical chain. In some embodiments, compounds of the invention
contain substituted amino selected from dimethylamino and diethylamino.

[0039] As used herein, "unsubstituted phenylalkynyl" refers to PhC/C--,
and "substituted phenylalkynyl" refers to phenylalkynyl containing one or
more substituents as described above.

[0040] As used herein, "nucleotide" refers to naturally occurring
nucleotides having naturally occurring nucleobases as well as
non-naturally occurring nucleotides that comprise monomer units
corresponding to the polynucleotides described above and/or a "modified
nucleobase". As used herein, the term "modified nucleobase" includes
nucleobases that differ from the naturally-occurring bases (e.g. A, G, C,
T and U) by addition and/or deletion of one or more functional groups,
differences in the heterocyclic ring structure (i.e., substitution of
carbon for a heteroatom, or vice versa), capable of nucleobase-specific
base-pairing with naturally occurring nucleobases. Examples of modified
nucleotides are those based on a pyrimidine structure or a purine
structure, with the former including, for example, 5-substituted
pyrimidines and the latter including, for example, 7-deazapurines and
their derivatives and pyrazolopyrimidines (see PCT WO 90/14353 and U.S.
Pat. No. 6,127,121).

[0044] As used herein, modified nucleotides do not encompass those wherein
the nucleobase has been modified by attachment of a blocking and/or
protecting group to an exocyclic nitrogen atom or to an endocyclic
nitrogen atom (i.e. --a nitrogen within the nucleobase ring) such that
the ability of the nucleotide to form a stable hydrogen bond is disrupted
or eliminated by the presence of the protecting group or blocking group,
see, for example, Will, S. C., et al. U.S. Pat. No. 6,001,611.

[0045] In some embodiments oligonucleotides of the present teachings can
be conjugated to at least one detectable label, nonfluorescent quencher
and/or at least one stabilizing moiety. In some embodiments
oligonucleotides of the present teachings that are conjugated to at least
one detectable label, nonfluorescent quencher and/or at least one
stabilizing moiety can serve as probes or primers.

[0046] The term "detectable label" refers to any moiety that, when
attached to oligonucleotides of the present teachings, render such
oligonucleotides detectable using known detection means. Exemplary
detectable labels include, but are not limited to, fluorophores,
chromophores, radioisotopes, spin-labels, enzyme labels or
chemiluminescent labels that allow for direct detection of a labeled
compound by a suitable detector, or a binding pair, for example, a
ligand, such as an antigen or biotin, that can bind specifically with
high affinity to a detectable anti-ligand, such as a labeled antibody or
avidin. In some embodiments the labels can be fluorescent dyes, such as
fluorescein or rhodamine dyes or fluorescent dye pairs, such as FRET
dyes.

[0047] In some embodiments, polynucleotides of the present teachings can
comprise one or more "nonfluorescent quencher" moieties. As used herein,
"nonfluorescent quencher" includes but is not limited to, for example,
particular azo dyes (such as DABCYL or DABSYL dyes and their structural
analogs), triarylmethane dyes such as malachite green or phenol red,
4',5'-diether substituted fluoresceins (U.S. Pat. No. 4,318,846),
asymmetric cyanine dye quenchers (see, Lee et al., U.S. Pat. No.
6,080,868 and Lee, et al., U.S. Pat. No. 6,348,596), or nonfluorescent
derivatives of 3- and/or 6-amino xanthene that is substituted at one or
more amino nitrogen atoms by an aromatic or heteroaromatic ring system
(Haugland, et al., U.S. Pat. No. 6,399,392).

[0048] "Nonfluorescent", as used herein, indicates that the fluorescence
efficiency of the quenching moiety in an assay solution as described for
any of the methods herein is less than or equal to 5 percent emission at
emission-λmax. In some embodiments, less than or equal to 1
percent emission at emission-λmax. In some embodiments of the
present teachings, the covalently bound quenching moiety exhibits a
quantum yield of less than about 0.1 percent emission at
emission-λmax. In some embodiments, less than about 0.01
percent emission at emission-λmax.

[0050] Suitable methods for attaching MGBs (as well as reporter groups
such as fluorophores and quenchers described above) through linkers to
polynucleotides are described in, for example, U.S. Pat. Nos. 5,512,677;
5,419,966; 5,696,251; 5,585,481; 5,942,610 and 5,736,626. Minor groove
binders include, for example, the trimer of
3-carbamoyl-1,2-dihydro-(3-H7)-pyrrolo[3,2-e]indole-7-carboxylate
(CDPI3) and the pentamer of N-methylpyrrole-4-carbox-2-amide
(MPC5). Additional MGB moieties are disclosed in U.S. Pat. No.
5,801,155. In certain embodiments, the MGBs can have attached water
solubility-enhancing groups (e.g., sugars or amino acids).

[0051] Polynucleotides of the present teachings can find use as primers
and/or probes in, for example, polynucleotide chain extension or ligation
reactions. As used herein, "chain extension reaction" refers to primer
extension reactions in which at least one polynucleotide of the present
teachings (e.g. --as primers or ligation probes) can be annealed to at
least one DNA template strand. After the step of annealing in a
polynucleotide chain extension reaction, the primer can then be extended
by at least one nucleotide to form an amplicon or extension product.
Alternatively, after the step of annealing in a polynucleotide chain
extension reaction, the primer can be ligated to a second ligation probe
to form a ligation product. The present teachings encompass all possible
chain extension reactions including, but not limited to, polymerase chain
reaction (PCR), nested PCR, asynchronous PCR, real time PCR, TaqMan
assays, DNA sequencing, cycled DNA sequencing, oligonucleotide ligation
assay (OLA), and fragment analysis, described in, for example, The PCR
Technique: DNA Sequencing II, Eaton Publishing Co. (1997), Genome
Analysis, A Laboratory Manual Volume 1: Analyzing DNA, Birren, B., Green,
E., Klapholz, S., Myers, R. M. and Roskams, J. Eds., Cold Spring Harbor
Laboratory Press (1997), Innis, M. et al., PCR Protocols: A Guide to
Methods and Applications, Academic Press (1989), Chen, C. et al., U.S.
Patent Application Pub. No. 2003/0207266 A1, Erlich, et al., U.S. Pat.
No. 5,314,809, U.S. Pat. No. 6,221,606 and Bi, W., et al., U.S. Pat. No.
6,511,810. Oligonucleotides of the present teachings can be used in any
of the above primer extension reactions as either primers or probes where
each is appropriate.

[0052] In some embodiments, the present teachings provide for methods of
primer extension comprising, annealing a polynucleotide primer to a
denatured DNA template such that, the polynucleotide primer anneals to a
complementary polynucleotide sequence on a strand of the denatured DNA
template to form a primer-template complex, and extending the primer
portion of the primer-template complex to form a double stranded
amplicon, wherein the polynucleotide primer is a primer according to the
present teachings.

[0053] In some embodiments, the present teachings provide for methods of
primer extension comprising, after the step of extending, denaturing the
double stranded amplicon. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least one time. In some
embodiments, the steps of annealing, extending and denaturing can be
repeated at least 10 times. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least 20 times. In some
embodiments, the steps of annealing, extending and denaturing can be
repeated at least 30 times. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least 40 times.

[0054] In some embodiments, the present teachings provide for a method of
primer extension comprising i) annealing a first polynucleotide primer
and a second polynucleotide primer to a first and second strand of a
denatured DNA template such that, the first polynucleotide primer anneals
to a complementary oligonucleotide sequence on the first strand of the
denatured DNA template and the second polynucleotide primer anneals to a
complementary oligonucleotide sequence on the second strand of the
denatured DNA template to form a first and a second primer-template
complex, and ii) extending the primer portion of at least one of the
first and second primer-template complex to form double stranded DNA
amplicon, wherein at least one of the first polynucleotide primer or the
second polynucleotide primer can be a polynucleotide of the present
teachings. In some embodiments, prior to the step of annealing, the
method can include the step of forming a mixture comprising a first
polynucleotide primer, a second polynucleotide primer, a DNA template,
and other primer extension reagents, including, for example buffers and
polymerases. In some embodiments, polymerases for use in the present
teachings can comprise at least one thermostable polymerase, including,
but not limited to, Taq, Pfu, Vent, Deep Vent, Pwo, UITma, and Tth
polymerase and enzymatically active mutants and variants thereof. Such
polymerases are well known and/or are commercially available.
Descriptions of polymerases can be found, among other places, at the
world wide web URL:
the-scientist.library.upenn.edu/yr1998/jan/profile1--980105.html.

[0055] In some embodiments, after the step of forming but prior to the
step of annealing, the method can include the step of denaturing the DNA
template to form a first strand of denatured DNA template and a second
denatured DNA template. In some embodiments, after the step of extending,
denaturing the double stranded DNA amplicon. In some embodiments, the
steps of annealing, extending and denaturing the double stranded DNA
amplicon can be repeated from 1-100 times. In some embodiments, the steps
of annealing, extending and denaturing the double stranded DNA amplicon
can be repeated from 10-100 times. In some embodiments, the steps of
annealing, extending and denaturing the double stranded DNA amplicon can
be repeated from 20-100 times. In some embodiments, the steps of
annealing, extending and denaturing the double stranded DNA amplicon can
be repeated from 30-100 times. In some embodiments, the steps of
annealing, extending and denaturing can be repeated at least one time. In
some embodiments, the steps of annealing, extending and denaturing can be
repeated at least 10 times. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least 20 times. In some
embodiments, the steps of annealing, extending and denaturing can be
repeated at least 30 times. In some embodiments, the steps of annealing,
extending and denaturing can be repeated at least 40 times.

[0056] In some embodiments, the present teachings provide for methods of
primer extension comprising, prior to the step of extending the primer
portion, annealing a polynucleotide probe to a first or second strand of
a denatured DNA template such that, the polynucleotide probe anneals to a
complementary polynucleotide sequence on the first strand of the
denatured DNA template and/or the polynucleotide probe anneals to a
complementary oligonucleotide sequence on the second strand of the
denatured DNA template. In some embodiments, the polynucleotide probe
comprises at least one detectable label. In some embodiments, the
polynucleotide probe further comprises at least one of a quencher, a
minor groove binder or both. In some embodiments, the polynucleotide
probe can be a polynucleotide of the present teachings.

[0057] In some embodiments, the present teachings provide "fragment
analysis" or "genetic analysis" methods, wherein labeled polynucleotide
fragments can be generated through template-directed enzymatic synthesis
using labeled primers or nucleotides, the fragments can be subjected to a
size-dependent separation process, e.g., electrophoresis or
chromatography; and, the separated fragments can be detected subsequent
to the separation, e.g., by laser-induced fluorescence. In some
embodiments, multiple classes of polynucleotides are separated
simultaneously and the different classes are distinguished by spectrally
resolvable labels.

[0058] In some embodiments, the present teachings provide a method of
fragment analysis in which fragment classes can be identified and defined
in terms of terminal nucleotides so that a correspondence is established
between the four possible terminal bases and the members of a set of
spectrally resolvable dyes. Such sets are readily assembled from the
fluorescent dyes known in the art by measuring emission and absorption
bandwidths using commercially available spectrophotometers. In some
embodiments, fragment classes are formed through chain termination
methods of DNA sequencing, i.e., dideoxy DNA sequencing, or Sanger-type
sequencing.

[0059] Sanger-type sequencing involves the synthesis of a DNA strand by a
DNA polymerase in vitro using a single-stranded or double-stranded DNA
template whose sequence is to be determined. Synthesis is initiated at a
defined site based on where an oligonucleotide primer anneals to the
template. The synthesis reaction is terminated by incorporation of a
nucleotide analog that will not support continued DNA elongation.
Exemplary chain-terminating nucleotide analogs include the
2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs) which lack the 3'-OH
group necessary for 3' to 5' DNA chain elongation. When proper
proportions of dNTPs (2'-deoxynucleoside 5'-triphosphates) and one of the
four ddNTPs are used, enzyme-catalyzed polymerization will be terminated
in a fraction of the population of chains at each site where the ddNTP is
incorporated. If labeled primers or labeled ddNTPs are used for each
reaction, the sequence information can be detected by fluorescence after
separation by high-resolution electrophoresis. In the chain termination
method, dyes of the invention can be attached to either sequencing
primers or dideoxynucleotides. In the method, fluorescent dye molecules
can be linked to a complementary functionality at, for example, the
5'-terminus of a primer, e.g. following the teaching in Fung, et al.,
U.S. Pat. No. 4,757,141; on the nucleobase of a primer; or on the
nucleobase of a dideoxynucleotide, e.g. via the alkynylamino linking
groups disclosed by Hobbs et al, in European Patent Application No.
87305844.0, and Hobbs et al., J. Org. Chem., 54: 3420 (1989) incorporated
herein by reference.

[0060] In some embodiments, labeled polynucleotides can be preferably
separated by electrophoretic procedures as disclosed in, for example,
Rickwood and Hames, Eds., Gel Electrophoresis of Nucleic Acids: A
Practical Approach, IRL Press Limited, London, 1981; Osterman, Methods of
Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag, Berlin, 1984;
or U.S. Pat. Nos. 5,374,527, 5,624,800 and/or 5,552,028. In some
embodiments, the type of electrophoretic matrix can be crosslinked or
uncrosslinked polyacrylamide having a concentration (weight to volume) of
between about 2-20 weight percent. In some embodiments, the
polyacrylamide concentration is between about 4-8 percent. In some
embodiments, for example in DNA sequencing, the electrophoresis matrix
can include a denaturing agent, e.g., urea, formamide, and the like.
Detailed procedures for constructing such matrices are given by, for
example, Maniatis et al., "Fractionation of Low Molecular Weight DNA and
RNA in Polyacrylamide Gels Containing 98% Formamide or 7 M Urea," in
Methods in Enzymology, 65: 299-305 (1980); Maniatis et al., "Chain Length
Determination of Small Double- and Single-Stranded DNA Molecules by
Polyacrylamide Gel Electrophoresis," Biochemistry, 14: 3787-3794 (1975);
Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York, pgs. 179-185 (1982); and ABI PRISM® 377
DNA Sequencer User's Manual, Rev. A, January 1995, Chapter 2 (Applied
Biosystems, Foster City, Calif.). It will be understood that optimal
electrophoresis conditions, for example, polymer concentration, pH,
temperature, and concentration of denaturing agent, employed in a
particular separation depends on many factors, including the size range
of the nucleic acids to be separated, their base compositions, whether
they are single stranded or double stranded, and the nature of the
classes for which information is sought by electrophoresis.

[0061] Subsequent to electrophoretic separation, labeled polynucleotides
can be detected by, for example, measuring the fluorescence emission from
a dye on the labeled polynucleotides. To perform such detection, the
labeled polynucleotides are illuminated by standard means, such as high
intensity mercury vapor lamps, lasers, or the like. In some embodiments,
the illumination means is a laser having an illumination beam at a
wavelength above about 600 nm. In some embodiments, the
dye-polynucleotides are illuminated by laser light generated by a He--Ne
gas laser or a solid-state diode laser. After illumination, fluorescence
intensity of the labeled polynucleotide can be measured by a
light-sensitive detector, such as a photomultiplier tube, charged coupled
device, or the like. Exemplary electrophoresis detection systems are
described elsewhere, e.g., U.S. Pat. Nos. 5,543,026; 5,274,240;
4,879,012; 5,091,652 and 4,811,218.

[0062] In some embodiments, the present teachings provide for a method of
fragment analysis comprising, annealing an polynucleotide primer to a
denatured DNA template, such that the polynucleotide primer anneals to a
complementary polynucleotide sequence on a strand of the denatured DNA
template to form a primer-template complex, extending the primer portion
of the primer-template complex in the presence of extendable nucleotide
triphosphates and non-extendable nucleotide triphosphates to form DNA
amplicon fragments and detecting the DNA amplicon fragments. In some
embodiments, the polynucleotide primer can be an oligonucleotide of the
present teachings. Further embodiments of fragment analysis methods in
accordance with the present teachings can be found in, for example, U.S.
Pat. No. 6,221,606 incorporated herein by reference.

[0063] As used herein, "ligation reaction" refers to reactions in which
allele specific ligation probes are annealed to at least one DNA template
strand to form a probe-template complex. After the step of annealing, a
covalent bond is then formed between the probe and the second
oligonucleotide fragment by a ligation agent to form a ligation product.
A ligation agent according to the present invention may comprise any
number of enzymatic or chemical (i.e., non-enzymatic) agents. For
example, ligase is an enzymatic ligation agent that, under appropriate
conditions, forms phosphodiester bonds between the 3'-OH and the
5'-phosphate of adjacent polynucleotides. Temperature-sensitive ligases,
include, but are not limited to, bacteriophage T4 ligase, bacteriophage
T7 ligase, and E. coli ligase. Thermostable ligases include, but are not
limited to, Taq ligase, Tth ligase, and Pfu ligase. Thermostable ligase
may be obtained from thermophilic or hyperthermophilic organisms,
including but not limited to, prokaryotic, eucaryotic, or archael
organisms. Some RNA ligases may also be employed in the methods of the
invention.

[0065] In some embodiments, the present teachings provide methods of
oligonucleotide ligation comprising, i) forming a complex comprising a
first and a second polynucleotide strand annealed to a DNA template such
that, the first polynucleotide strand anneals to a first complementary
polynucleotide sequence on the strand of the denatured DNA template and
the second polynucleotide strand anneals to a second complementary
polynucleotide sequence on the strand of the denatured DNA template,
wherein the second complementary polynucleotide sequence on the strand of
the denatured DNA template is located 5' to the first complementary
polynucleotide sequence on the strand of the denatured DNA template, and
ii) forming a stable covalent bond between the first and second
polynucleotide strands, wherein at least one of the first polynucleotide
strand or the second polynucleotide strand is a polynucleotide of the
present teachings.

[0066] In some embodiments, the present teachings provide a method of
oligonucleotide ligation comprising, annealing a first oligonucleotide to
a strand of a denatured DNA template such that, the first oligonucleotide
anneals to a complementary oligonucleotide sequence on the strand of the
denatured DNA template to form a first oligonucleotide-template complex,
annealing a second oligonucleotide to an oligonucleotide sequence on the
first oligonucleotide-template complex that is complementary to the
second oligonucleotide to form a second oligonucleotide-template complex,
wherein the second oligonucleotide anneals to the first
oligonucleotide-template complex so that the 3'-terminus of the first
oligonucleotide and the 5'-terminus of the second oligonucleotide are
associated with adjacent nucleotides of the denatured DNA template, and
forming a stable covalent bond between the 3'-terminus of the first
oligonucleotide and the 5'-terminus of the second oligonucleotide. In
some embodiments, at least one of the first oligonucleotide or the second
oligonucleotide can be an oligonucleotide of the present teachings.

[0067] In some embodiments, the present teachings provide for a method for
detecting a target polynucleotide sequence comprising (a) reacting a
target polynucleotide strand and a target-complementary strand with a
first probe pair and a second probe pair, the first probe pair comprising
(i) a first polynucleotide probe containing a sequence that is
complementary to a first target region in the target strand and (ii) a
second polynucleotide probe comprising a sequence that is complementary
to a second target region in the target strand, wherein the second region
is located 5' to the first region and overlaps the first region by at
least one nucleotide base, and the second probe pair comprising (i) a
third polynucleotide probe containing a sequence that is complementary to
a first region in the target-complementary strand and (ii) a fourth
polynucleotide probe containing a sequence that is complementary to a
second region in the target-complementary strand, wherein the second
region is located 5' to the first region and overlaps the first region by
at least one nucleotide base, under conditions effective for the first
and second probes to hybridize to the first and second regions in the
target strand, respectively, forming a first hybridization complex, and
for the third and fourth probes to hybridize to the first and second
regions in the target-complementary strand, respectively, forming a
second hybridization complex, (b) cleaving the second probe in the first
hybridization complex, and the fourth probe in the second hybridization
complex, to form (i) a third hybridization complex comprising the target
strand, the first probe, and a first fragment of the second probe having
a 5' terminal nucleotide located immediately contiguous to a 3' terminal
nucleotide of the first probe, and (ii) a fourth hybridization complex
comprising the target-complementary strand, the third probe, and a first
fragment of the fourth probe having a 5' terminal nucleotide located
immediately contiguous to a 3' terminal nucleotide of the third probe,
(c) ligating the first probe to the hybridized fragment of the second
probe to form a first ligated strand hybridized to the target strand, and
ligating the third probe to the fragment of the fourth probe to form a
second ligated strand hybridized to the target-complementary strand, (d)
denaturing the first ligated strand from the target strand and the second
ligated strand from the target-complementary strand, and (e) performing
one or more additional cycles of steps (a) through (d), with the proviso
that in the last cycle, step (d) is optionally omitted.

[0068] In some embodiments, the first region can overlap the second region
by one nucleotide base. In some embodiments, the 5' ends of the first and
third probes can terminate with a group other than a nucleotide 5'
phosphate group. In some embodiments, the 5' ends of the first and third
probes can terminate with a nucleotide 5' hydroxyl group. In some
embodiments, the 5' ends of the second and fourth probes can terminate
with a group other than a nucleotide 5' phosphate group. In some
embodiments, the 5' ends of the second and fourth probes can terminate
with a nucleotide 5' hydroxyl group. In some embodiments, the 5' ends of
the first, second, third and fourth probes can each independently
terminate with a group other than a nucleotide 5' phosphate group. In
some embodiments, the 3' ends of the second and fourth probes can each
independently terminate with a group other than a nucleotide 3' hydroxyl
group. In some embodiments, the 3' ends of the second and fourth probes
can terminate with a nucleotide 3' phosphate group.

[0069] In some embodiments, at least one of the probes can contain a
detectable label. In some embodiments, the label can be a fluorescent
label. In some embodiments, the label can be a radiolabel. In some
embodiments, the label can be a chemiluminescent label. In some
embodiments, the label can be an enzyme. In some embodiments, at least
one of the first probe and the third probe can contain a detectable
label. In some embodiments, each of the first probe and third probe can
contain a detectable label. In some embodiments, the detectable labels on
the first probe and third probe can be the same. In some embodiments, at
least one of the second probe and the fourth probe can contain a
detectable label. In some embodiments, each of the second probe and the
fourth probe contains a detectable label. In some embodiments, the second
probe and fourth probe can contain the same detectable label.

[0070] In some embodiments, the step of cleaving produces a second
fragment from the second probe which does not associate with the third
hybridization complex, and the method further includes detecting said
second fragment.

[0071] In some embodiments, at least one of the second probe and the
fourth probe contains both (i) a fluorescent dye and (ii) a quencher dye
which is capable of quenching fluorescence emission from the fluorescent
dye when the fluorescent dye is subjected to fluorescence excitation
energy, and said cleaving severs a covalent linkage between the
fluorescent dye and the quencher dye in the second probe and/or fourth
probe, thereby increasing an observable fluorescence signal from the
fluorescent dye. In some embodiments, the second probe and the fourth
probe can each contain (i) a fluorescent dye and (ii) a quencher dye.

[0072] In some embodiments, the step of cleaving further produces a second
fragment from the fourth probe that does not associate with the fourth
hybridization complex, and the method further includes detecting both
second fragments. In some embodiments, the second fragment comprises one
or more contiguous nucleotides that are substantially non-complementary
to the target strand. In some embodiments, one or more contiguous
nucleotides comprise 1 to 20 nucleotides.

[0073] In some embodiments, the method further includes immobilizing the
second fragment on a solid support. In some embodiments, the method
further includes subjecting the second fragment to electrophoresis. In
some embodiments, the method further includes detecting the second
fragment by mass spectrometry. In some embodiments, the method further
comprises detecting the second fragment after the last cycle. In some
embodiments, the method further comprises detecting the second fragment
during or after a plurality of cycles. In some embodiments, the method
further comprises detecting the second fragment during all of the cycles.
In some embodiments, the method further includes detecting the first
hybridization complex, the second hybridization complex, or both, after
at least one cycle. In some embodiments, the method further includes
detecting the third hybridization complex, the fourth hybridization
complex, or both, after at least one cycle. In some embodiments, the
method further includes detecting the first ligated strand, the second
ligated strand, or both, after at least one cycle. In some embodiments,
said detecting comprises an electrophoretic separation step.

[0074] Further embodiments of the ligation method for detecting a target
polynucleotide can be found in Bi, W., et al., U.S. Pat. No. 6,511,810
incorporated herein by reference in its entirety.

EXAMPLES

Materials and Methods

[0075] Unless otherwise indicated, all synthesis reactions were carried
out in oven or flame dried glassware, under an atmosphere of argon.
Tetrahydrofuran (THF) and methylene chloride (CH2Cl2) were
distilled from calcium hydride (CaH2) under Argon. Unless otherwise
indicated, all other solvents were used as received from the distributor.
Thin layer chromatography (TLC) was performed on 1 mm silica gel plates
purchased from Sigma-Aldrich (Milwaukee, Wis.) and visualized with UV
light (Spectroline; model ENF-240C) or stained with KMnO4 or
phosphomolybdic acid. Flash column chromatography was performed using
silica gel with an average particle size of 40 μm purchased from
Sigma-Aldrich (Milwaukee, Wis.). 8-Aza-7-deaza-2'-deoxyguanine was
purchased from EPOCH Biosciences Inc (Bothell, Wash.).
8-Aza-7-deaza-2'-deoxyadenosine was purchased from Berry & Associates Inc
(Dexter, Mich.). Non-derivatized CPG support was obtained from Applied
Biosystems Inc (P/N 360139, Foster City, Calif.). Unless otherwise
indicated, all other reagents were purchased from Sigma-Aldrich. Unless
otherwise indicated, automated DNA synthesis was carried out on an ABI
394 DNA synthesizer at a 0.2 μmol scale following the standard
protocol. Oligonucleotides were purified by reversed phase HPLC on an
Agilent 1100 HPLC system. ESI-TOF mass spectra were recorded on a
MARINER® Biospectrometry Workstation (Applied Biosystems, Foster
City). The purity of synthesized oligo-nucleotides was checked by
capillary electrophoresis (CE) on an Agilent CE system.

Oligonucleotide Synthesis

8-Aza-7-deaza-2'-deoxyadenosine CPG

[0076] 8-Aza-7-deaza-2'-deoxyadenosine CPG was prepared according to
Scheme 3.

##STR00010##

N-Benzoyl-5'-dimethoxytrityl-2'-deoxyadenosine

[0077] To a stirred solution of 50 mg of 8-aza-7-deaza-2'-deoxyadenosine
in 1 mL of dry pyridine was added 100 mg of DMT-Cl, 42 μL Et3N
and 600 μg DMAP. The reaction was stirred at room temperature for 4
hours and then 128 μL of trimethylsilyl chloride (TMS-Cl) was added.
The reaction was then stirred for an additional 30 minutes at room
temperature at which time 116 μL of benzoyl chloride (BzCl) was added
and the mixture stirred for an additional 2 hours. The mixture was then
cooled to 0° C. in an ice bath and 200 μL of water was added.
After stifling for 5 minutes, 400 μL of 29% aqueous ammonia was added
and the mixture was stirred at room temperature for 30 minutes. The
solvent was evaporated and the residue was partitioned between 3 mL of
CH2Cl2 and 3 mL of 5% aqueous NaHCO3. The layers were
separated and organic layer was evaporated to dryness. The crude product
was purified by silica gel chromatography (12.5/1 CH2Cl2/MeOH)
to give 16 mg of the desired product (12% yield).

[0078] To a stirred solution of 16 mg of
N-benzoyl-5'-dimethoxytrityl-2'-deoxy-adenosine in 2 mL of
CH2Cl2 was added 4.3 mg succinic anhydride, 1.5 mg DMAP and 7.2
μL Et3N. The reaction was stirred at room temperature for 12
hours at which time the reaction was diluted with 10 mL of 5% aqueous
citric acid. The organic layer was washed with sat. NaCl, dried over
anhydrous Na2SO4 and the solvent removed under vacuum. The
crude product was purified by silica gel chromatography (100/50/1
CH2Cl2/MeOH/Et3N) to give 12 mg of the desired product.

8-Aza-7-deaza-2'-deoxyadenosine CPG

[0079] To a solution of 12 mg
N-benzoyl-3'-(3-carboxypropionyl)-5'-dimethoxytrityl-2'-deoxyadenosine in
1 mL of dry DMF was added 3.6 μL DIPEA, 5.3 mg of HBTU and 300 mg of
CPG. The mixture was stirred for 2 hours at room temperature and then
washed with DMF followed by THF. The 8-Aza-7-deaza-2'-deoxyadenosine CPG
product was dried under high vacuum.

[0081] 8-Aza-7-deaza-2'-deoxyguanisine CPG was prepared according to
Scheme 4 below.

##STR00011##

[0082] To a stirred solution of
N2-(N,N-Dimethylformamido-3'-(3-carboxypropionyl)-5'-dimethoxytrityl-8-az-
a-7-deaza-2'-deoxyguanosine in 10 mL of dry DMF was added 117 μL of
DIPEA, 86 mg of HBTU, and 5 g of CPG. The mixture was stirred for 4 hours
at room temperature and then washed with DMF, CH3CN and
CH2Cl2. The 8-aza-7-deaza-2'-deoxyguanosine CPG product was
dried under high vacuum.

3'-Terminal oligonucleotides of 8-aza-7-deaza-2'-deoxyguanosine and
8-aza-7-deaza-2'-deoxyadenosine

[0094] The progress of all PCR reactions was monitored in real time on an
ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems;
Foster City, Calif.) as described in SYBR® Green PCR Master Mix and
RT-PCR: Protocol (Applied Biosystems, 2002).

[0095] Thermal Cycling:

[0096] Thermal Cycling Protocol #1:

[0097] Amplification temperature cycling was carried out using the
following thermal cycling protocol.

[0105] Amplification reactions using 2'-deoxy-PPA 3'-terminal primers,
shown in Table 1, were carried out according to Thermal Cycling Protocol
#2.

[0106] For each primer pair, amplification reactions using unmodified and
3'-modified primers were run with gDNA corresponding to the primer pair.
Additionally, amplification reactions were also run without any template
as no template control (NTC) reactions.

[0107] Gel electrophoresis analysis was carried out as described above.
Gel electrophoresis showed a significant amount of non-specific
amplification product at about 50 bp in NTC amplifications with
unmodified primers indicating the formation of primer-dimer amplicons. On
the other hand, decreased primer-dimer amplicon formation was observed in
gel electrophoresis images of NTC amplifications using 3'-modified
primers. Gel electrophoresis of gDNA template amplification reactions
using unmodified primers clearly showed a band corresponding to the
desired template amplicon and also showed a band at corresponding to
primer-dimer amplicon formation.

[0108] Amplification of AGT template using unmodified primers clearly
showed a band at about 75 bp corresponding to the desired template
amplicon and also showed a band at about 50 bp corresponding to
primer-dimer amplicon formation. On the other hand, amplification of AGT
template using 3'-modified primers clearly showed a band at about 75 bp
corresponding to the desired template amplicon but also showed no
primer-dimer amplicon formation.

[0109] Real time PCR monitoring showed that the gDNA amplification
efficiencies using unmodified- and 3'-modified-primers were very similar.
On the other hand, while the NTC amplification using unmodified primers
gave a Ct value, which is the calculated cycle number at which
detectable signal rises above background, of about 28, NTC amplification
using ppA modified primers did not give a measurable signal until about
the 33rd cycle.

[0110] Amplification of ATIR template using unmodified primers clearly
showed a band at about 200 bp corresponding to the desired template
amplicon and also showed a band at about 50 bp corresponding to
primer-dimer amplicon formation. On the other hand, amplification of ATIR
template using 3'-modified primers clearly showed a band at about 200 bp
corresponding to the desired template amplicon but also showed no
primer-dimer amplicon formation.

[0111] Real time PCR monitoring showed that the gDNA amplification
efficiencies using unmodified- and 3'-modified-primers were good. NTC
amplification using unmodified primers gave a Ct value of about 33,
whereas NTC amplification using ppA modified primers did not give a
measurable signal until about the 42nd cycle.

2'-deoxy-PPG 3'-Terminal Primers

[0112] Amplification reactions using 2'-deoxy-PPG 3'-terminal primers,
shown in Table 2, were carried out according to Thermal Cycling Protocol
#1.

[0113] For each primer pair, amplification reactions using unmodified and
3'-modified primers were run with gDNA corresponding to the primer pair.
Additionally, amplification reactions using unmodified and 3'-modified
primers were run without any template as no template control (NTC)
reactions.

[0114] Gel electrophoresis analysis was carried out as described above.
Gel electrophoresis showed a significant amount of non-specific
amplification product at about 50 bp in NTC amplifications with
unmodified primers indicating the formation of primer-dimer amplicons. On
the other hand, significantly decreased primer-dimer amplicon formation
was observed in gel electrophoresis images of NTC amplifications using
3'-modified primers. Gel electrophoresis of gDNA template amplification
reactions using unmodified primers clearly showed a band corresponding to
the desired template amplicon and also showed a band at corresponding to
primer-dimer amplicon formation.

[0115] Amplification of AGT template using unmodified primers clearly
showed a band at about 75 bp corresponding to the desired template
amplicon and also showed a band at about 50 bp corresponding to
primer-dimer amplicon formation. On the other hand, amplification of AGT
template using 3'-modified primers clearly showed a band at about 75 bp
corresponding to the desired template amplicon but also showed no
primer-dimer amplicon formation.

[0116] Real time PCR monitoring showed that the gDNA amplification
efficiencies using unmodified- and 3'-modified-primers were very similar.
On the other hand, while the NTC amplification using unmodified primers
gave a Ct value of about 23, a value which was almost equivalent to
the gDNA amplifications, NTC amplification using ppG modified primers did
not give a measurable signal until after the 33rd cycle.

[0117] Amplification of ATIR template using unmodified primers clearly
showed a band at about 200 bp corresponding to the desired template
amplicon and also showed a band at about 50 bp corresponding to
primer-dimer amplicon formation. On the other hand, amplification of ATIR
template using 3'-modified primers clearly showed a band at about 200 bp
corresponding to the desired template amplicon but also showed no
primer-dimer amplicon formation.

[0118] Real time PCR monitoring showed that the gDNA amplification
efficiencies using unmodified- and 3'-modified-primers were similar. On
the other hand, while the NTC amplification using unmodified primers gave
a Ct value of about 22, NTC amplification using ppG modified primers
did not give a measurable signal until about the 30th cycle.

[0119] λ-DNA Amplifications:

[0120] To test whether 3'-modified primers of the present teachings could
reduce non-specific amplification without effecting amplification
efficiency of longer amplicons, λ-DNA was used as a template with
primers designed to make a 1700 bp target amplicon.

[0121] 2'-Deoxy-PPG λ-DNA Amplification:

[0122] Amplification reactions using 2'-deoxy-PPG containing primers,
shown in Table 3, were carried out according to Thermal Cycling Protocol
#3. As above, both real time analysis and gel electrophoresis showed that
even with longer amplicons, 3'-modification with 2'-deoxy-PPG resulted in
significant reduction in primer-dimer amplification without adversely
effecting target amplification efficiency. Amplification efficiencies of
λ-DNA template using both unmodified and modified primers were
nearly identical, Ct about 24. NTC amplification using unmodified
primers gave a Ct value of about 32, whereas NTC amplification using
2'-deoxy-PPG modified primers gave no detectable signal until after the
42nd cycle.